The importance of p53 as a tumor suppressor is well established. However, because p53 affects the functioning of so many fundamental cellular processes in its capacity as a transcription factor and through numerous protein-protein interactions, dissecting the mechanisms of p53 action remains challenging. Approximately 30 different sites on p53 can be post-translationally modified by phosphorylation, acetylation, methylation, ubiquitinylation, neddylation or sumoylation. Post-translational modifications of p53 can affect its stability, its activity as a transcription factor and its interactions with specific proteins. Much recent work on the transcriptional activating function of p53 has focused on its ability to recruit histone acetyl transferase co-activators such as CBP, 300 and PCAF to the promoters of specific target genes. p53 can also activate transcription through direct interaction with components of the transcription complex. We recently characterized the complex formed between an N-terminal transactivation domain (TAD) peptide of p53 and a component of the human or yeast general transcription factor II H (TFIIH) by physical methods. We found that the pleckstrin homology (PH) domain of the p62 (human) or Tfb1 (yeast) subunits specifically bound p5320-73, corresponding to the second transactivation subdomain, TAD2. Using NMR, we mapped chemical shift changes induced by complex formation onto the solution structures of the p62 or Tfb1 PH domains. Phosphorylation of Ser46 or Thr55 within the p53 TAD2 resulted in substantially tighter binding to the p62 PH domain. P53 can be phosphorylated at Ser46 by p38 mitogen activated protein kinase (MAPK) or by homeodomain interacting protein kinase (HIPK) and at Thr55 by TATA-associated factor 1 (TAF1) in response to various stimuli. When Ser46 is phosphorylated, the strengthened binding between p53 TAD2 and p62 may contribute to the increased induction of target genes such as LRDD or PPM1D in which the binding site for p53 is located very close to the start of transcription. This work suggests a mechanistic link between a post-translational modification of a specific site on p53 and specific functional effects. In addition, we have been engaged in a systematic investigation of the effects of particular sites of post-translational modification in p53 through the generation of "knock-in" mice. In these mice, both alleles of the genomic p53 sequence have been mutated to remove a site of potential post-translational modification, which allows the loss of a specific site of post-translational modification to be examined in the context of the whole organism. In earlier work, we reported that changing the murine p53 Ser18 (the equivalent of human Ser15) to alanine resulted in reduced transactivation or reduced repression of target genes, and impaired apoptosis and cell cycle arrest. The p53S18A mice, however, were not predisposed to spontaneous tumerogenesis. Recently, we reported the effects of changing the mouse p53 Lys317 (the equivalent of human K320) to arginine, thereby preventing lysine acetylation at that site. We found that, although DNA damage-dependent cell cycle arrest in murine embryonic fibroblasts was not affected by the K317R mutation, DNA damage-induced apoptosis was substantially reduced in thymocytes, crypts of the small intestine and the developing retina. Furthermore, following exposure to ionizing radiation, the mRNA levels of some p53 target genes, such as the pro-apoptotic genes Bbc3, Phlda3 and Pmaip1, were significantly higher in K317R thymocytes than in wild-type thymocytes, whereas the induction of other p53 target genes was not significantly different. These results suggest that acetylation of lysine 317 contributes to cell or tissue-specific tuning of the p53 response. We have now extended this approach to examine the effects of changing both Ser18 and Ser23 of mouse p53 (equivalent to human Ser15 and Ser20) to alanine.